The present invention relates generally to solid phase processing and, more particularly to a column for use in solid phase processing including solid phase synthesis and purification of complex chemicals such as oligonucleotides and the like.
A variety of separative, synthetic, and enzymatic or otherwise catalytic processes use beds of particulate material with transport of reactants, reagents and products or eluants in solution through the bed. In addition, many reactions are known in which the products are separated by concentration in one of two or more phases. These processes include, among others, ion exchange chromatography, gel filtration, ion exclusion chromatography, affinity chromatography, separations based on hydrophobicity, purification based on hybridization, peptide synthesis, oligonucleotide synthesis, and polysaccharide synthesis including combinations of the last three. These processes may be carried out on a small scale for analytical purposes or process design, and are then often scaled up for preparative work. In nearly all examples the solid phase particulates are packed in a closed column with a porous frit on the lower end, an optional frit at the top, and with fluid-connections at both ends so that liquid can flow in either direction through the bed. To achieve efficiency and high resolution with solid phase supports, all volume elements of all fluids should flow through paths of identical composition and nearly identical length, and all particles in the bed should be exposed to the same succession of liquids under the same conditions.
In solid phase systems, some interaction occurs between the solutes run through the bed and the particles composing the bed. This interaction may be based on secondary forces (ionic, hydrophobic, or on immunochemical interactions, or base pairing) or primary valencies as when amino acids or nucleotides are added to a growing chain on the solid phase support, or when immobilized enzymes cleave substrates flowing through the bed, or when enzymes in solution react with substrates attached to the packing. In addition, solvents or reagents of successively differing composition which dissociate adsorbed or otherwise attached bound molecular species, or which cleave off protective groups, or compounds including polymers which have been synthesized on the support may be made to flow through the support. The dissociated or cleaved substances then are free to flow out of the bed in flowing liquid.
In particular, nucleic acid synthesis (generally referred to as xe2x80x9cDNA synthesisxe2x80x9d) is the process of constructing synthetic single-stranded oligonucleotide through linking of nucleotide, the basic building blocks for DNA. In an automated system, the various steps are carried out by a reagent delivery system which dispenses a number of chemical reagents in a predetermined sequence in a cycle into a synthesis reaction column containing the solid-phase support, according to instructions from the system controller or computer. After the desired number of cycles have been completed, the synthesized oligonucleotide is separated from the reaction column and collected in a vial. This step is generally referred to as xe2x80x9ccleavagexe2x80x9d. The oligonucleotide may further be subject to a step generally referred to as xe2x80x9cdeprotectionxe2x80x9d to complete isolation of the oligonucleotide. In a process for synthesizing polynucleotides on a solid support, the solid support traditionally consists of glass beads of controlled porosity (CPG) or, more generally, of particles of a functionalized inorganic or organic polymer.
The isolation of oligonucleotide involves the treatment of the solid bound oligonucleotide with a cleavage and/or deprotection reagent. Typically, this reagent is concentrated ammonia solution in water but can be other homogeneous or heterogeneous solutions of appropriate bases, alcohols and water. The cleavage and deprotection process is typically performed in two steps. The cleavage of the oligonucleotide is performed at room temperature for approximately one hour before decanting the mixture into a pressure-sealable vessel for extended higher temperature treatment to effect the removal of secondary protecting groups on the synthetic oligonucleotide. This two step process reduces the quantity of support related contaminants in the final isolated product.
The present invention is directed to an improved column for use in solid phase synthesis or purification. In a specific embodiment, the column is a disposable plastic column for use in solid phase synthesis or purification of complex chemicals, such as biomolecules and more specifically oligonucleotides.
In some embodiments, the column has a top orifice with a sufficient diameter so that a fluid line or a multiple fluid line bundle may dispense fluids into the column with great efficiency. The column has an upper cavity portion configured and sized to render it compatible with dispensing pippetors, so that it can be used as a pipette tip or a pipettor can be used to aspirate the column. The column has a lower cavity portion with a shoulder for ready placement of a lower frit to contain the solid support in a central cavity portion of the column which has a uniform cross-section. An upper frit can be conveniently placed in the uniform central cavity portion to seal the solid phase resin. The lower end tip of the column is configured as a luer-type fitting to provide a male luer connection. The upper cavity portion is configured to interface with the male luer of another column, so that two or more columns to be connected conveniently in series.
In accordance with an aspect of the present invention, a column for solid phase processing comprises a housing including a cavity extending from a top orifice at an upper end to a bottom orifice at a lower end which is smaller than the top orifice at the upper end. The cavity decreases in cross-sectional size from the top orifice at the upper end to the bottom orifice at the lower end. The lower end of the housing is configured as a male luer.
In some embodiments, the cavity of the housing includes an upper cavity portion adjacent the top orifice, a central cavity portion which is smaller in cross-section than the upper cavity portion, and a lower cavity portion which is smaller in cross-section than the central cavity portion and adjacent the bottom orifice. In specific embodiments, the top orifice is at least about 5 mm in diameter. The top orifice may be at most about 9 mm in diameter. The cavity is typically circular in cross-section.
In accordance with another aspect of the present invention, a column for solid phase processing comprises a housing including a cavity extending from a top orifice at an upper end to a bottom orifice at a lower end which is smaller than the top orifice at the upper end. The cavity decreases in cross-sectional size from the top orifice at the upper end to the bottom orifice at the lower end. The lower cavity portion includes a shoulder to support a lower frit for containing a solid support to occupy a portion of the central cavity portion. The central cavity portion has a uniform cross-section for receiving an upper frit to seal the solid support.
In accordance with another aspect of the invention, a column for solid phase processing comprises a housing including a cavity extending from a top orifice at an upper end to a bottom orifice at a lower end which is smaller than the top orifice at the upper end. The cavity includes an upper cavity portion adjacent the top orifice, a lower cavity portion adjacent the bottom orifice, and a central cavity portion between the upper cavity portion and the lower cavity portion. The lower end of the housing is configured as a male luer. The upper cavity portion is shaped to receive a male luer at a lower end of a housing of another column for connecting the columns in series.
In some embodiments, the upper cavity portion is tapered inward decreasing in cross-sectional size from the top orifice.